As traditionally conducted, a set of adult blood tests necessitates drawing whole blood with 3 to 6 of the well-known pre-evacuated blood collection tubes (e.g. Vacutainer™, Becton Dickinson and Company, East Rutherford, N.J.), each with typically 2 to 10 milliliter capacity. Plasma or serum is typically obtained when whole blood collected in this fashion is processed by centrifuging or filtering, performed within minutes from the sample being drawn unless a stabilizing substance has been added to permit delayed separation.
The availability of sensitive biological assays has also made it possible to run accurate tests employing much smaller sample volumes than previously employed. For instance, multiple tests are available that can be performed employing less than 0.1 milliliter of the fluid, using bio-array techniques. No very simple, inexpensive and rapidly operable device has been commercially available for providing serum or plasma extraction at this size volume.
Typical delays in obtaining plasma or serum can range from 10 minutes when a centrifuge is on site to over one hour when it is within the facilities. The delay can be days if samples must be transported to remote locations. These delays defeat the value of onsite diagnostics made possible by the new bio-array (biochip) technologies. The major benefit of biochip technology is to offer a diagnosis within 15 to 60 minutes, saving critical time for intervention as well as saving costs.
Small volume whole blood collection, per se, however, has long been available. It was originally developed for blood tests for infants and small animals. For this purpose, evacuated collection tubes have been available for drawing a fraction of a milliliter or a few milliliters of blood. (Extremely small blood volumes have also traditionally been obtained by use of a puncture wound. The finger for instance is pricked with a lancet and then squeezed until a fluid drop of, for example, 10-20 microliters is obtained).
In general, current methods for achieving small volumes of serum from whole blood typically involve numerous steps and employ multiple pieces of equipment and disposable items. Kits are available for these purposes from many sources, examples being: Unopette® (Becton Dickinson and Company); Fisherbrand® microhematocrit and capillary tubes (Fisher Scientific Company, Hampton N.H.); and StatSampler® capillary blood collection kit (StatSpin, Norwood, Mass.). Each of these relies on multiple separate components for performing the functions of sample collection, processing, and recovery.
There have been many attempts to develop more convenient devices, but no reliable, simple and simply-operated hand-held filtering device is available that can produce hemolysis-free serum or plasma.
Prior art in the general field include U.S. Pat. Nos. 2,460,641; 3,814,258; 4,343,705; 4,477,575; 4,540,492; 4,828,716; 4,883,068; 4,906,375; 4,960,130; 5,030,341; 5,181,940; 5,308,508; 5,364,533; 5,413,246; 5,471,994; 5,555,920; 5,681,529; 5,683,355; 5,759,866; 5,876,605; 5,919,356; 5,979,669; 5,996,811; 6,045,699; 6,170,671; 6,261,721; 6,225,130; 6,406,671; 6,410,334; 6,465,256; 6,471,069; 6,479,298; 6,497,325; 6,506,167; 6,516,953; 6,537,503; 6,659,288; 6,659,975; 6,755,802; 6,803,022; 6,821,789; 7,070,721; 7,153,477; 7,767,466; 7,744,820; 7,927,810; and 7,993,847; and US 2010/0093551.
It is recognized to be desirable to work quickly and efficiently with blood samples of the order of 1 milliliter volume. Most protein analyzers for instance require 10 to 100 micro-liters per test and it is common to employ 10 or so tests. Multiplexed biomarker cassettes, e.g. those employing micro arrays, typically run 8 to 12 assays simultaneously, and call for less than 100 micro-liter of serum or plasma for the set of assays.
Devices and techniques made possible by the present disclosure can simply, inexpensively and rapidly meet the need for obtaining suitable blood serum and other blood-derived fluids from small volume whole blood samples. Neither centrifuge separation nor other inconvenient techniques are employed, while sterile separation at point of collection or point of patient treatment can be achieved.
The level of hemolysis, the presence of hemoglobin within the plasma or serum as a result of cell damage, may not interfere with most diagnostic tests and specifically most protein or ELISA tests, but excess hemolysis could be indicative of patient health conditions that would need to be considered, and consequently lead to an erroneous diagnosis. More specifically the presence of hemoglobin in serum may yield erroneous reading of the blood potassium concentration. For these reasons desirable hemolysis quantifications of low value have been established.
Consequently, in order to be practical, a plasma or serum extraction processor device needs to keep damage to red cells to a minimum.
It is important to consider further that the venous puncture commonly causes some red cells breakage so that the standards that have been established to define levels of acceptable hemolysis leave little room for additional hemolysis by serum separation features. This is where previous devices have failed to meet exacting standards.
U.S. Pat. No. 4,477,575 teaches the use of glass fibers with diameter from 1 to 4 micron can be efficiently used to separate cells from plasma or serum in a depressurization syringe-like device. The use of this type of glass fiber has been adopted in later processes as well as the suction/depressurization serum extraction method, as exemplified by U.S. Pat. No. 5,364,533 that employs pre-evacuation of a device.
Later prior art as exemplified in U.S. Pat. Nos. 7,744,820, 7,927,810 and 7,993,847 and US 2007/0082370 describe blood collection and serum separation using a an internal negative pressure plurality of interconnected tubes as well as the use of glass fibers as filter medium. This prior art attempts to control hemolysis by stratification of filtration porosity using a membrane with a void ratio under 30% and/or altered retention properties of the filtration column media.
U.S. Pat. No. 5,876,605 similarly uses glass fiber and seeks to minimize hemolysis with suitable mixing of the blood with an aqueous solution.
U.S. Pat. Nos. 5,979,669, 5,996,811, 6,045,699 and 6,170,671 also use glass fiber as a filtrate material and incorporate means to regulate outflow of filtrate in order to accommodate variation in hematocrit and control hemolysis. They all show how a number of interconnected tubular devices create a pressure difference by connection to a suction pump or device. Typically the final outlet filter membrane is constructed to regulate serum outlet flow.
U.S. Pat. No. 5,979,669 teaches “In another aspect of the blood filter unit of the invention, a flow area-regulating member is provided on the blood filtering material on the filtrate outlet side which is, in general, the microporous membrane. The flow area-regulating member is made of liquid-impermeable material, and has an opening having an area smaller than the blood filtering material thereby regulates so that filtrate flows out through the opening. A suitable area of the opening is about 20 to 90%, preferably about 50 to 90% of the blood filtering material area on the filtrate outlet side.”
“The flow area-regulating member can be made by various commercial adhesive tapes, plastic film, thin plastic sheet or the like, and adhesive may be applied to the adhering face of the blood filtering material.”
U.S. Pat. Nos. 5,364,533 and 5,979,669 teach the use of a number of interconnected and detachable successions of tubes to create a pressure difference across a filter assembly in order to obtain plasma by filtration.
U.S. Pat. Nos. 6,506,167, 6,659,288 and 6,045,699 suggest the use of stratified filtration column as well as external active sequencing of controlled differential pressure forcing the blood through the filter column or the entire device from blood inlet to filtrate outlet.
U.S. Pat. No. 6,045,699 teaches that a suitably hemolysis-free filter device can be constructed where pressure differential across a filter assembly of an evacuated device is actively controlled from a tethered pressure source external to the filter device. It teaches to sequence the pressure differential with a pressure sequencer where filtration begins with a low pressure differential which is “controllably increased” as filtration progresses. The patent teaches using active external equipment such as a peristaltic pump or a syringe. It teaches to “trace” pressure different variation with time and to “adjust suction or pressurizing speed.”
U.S. Pat. No. 7,993,847 teaches the use of filter assembly in which a membrane exit filter, in a passive manner, regulates the pressure differential across a filter assembly, seeking to yield a substantially hemolysis-free serum sample.
The membrane exit filter has a number of micron size apertures. But such a membrane is totally ineffective to limit the flow of air across it as air molecules are sub angstrom in dimensions. Such a membrane is effective only to limit liquid flow and have any effect much later in the filtration process when blood has already reached and serum or plasma has already travelled through the filter assembly. Such a device starts the filtration process with maximum pressure differential across the filter assembly and is insufficient to control hemolysis to the low level necessary.
A prior attempt by one of us to meet the present need is shown in US2010/0093551. It has the requirement of repeated hand movements and other drawbacks, and lacks the critical flow rate or pressure differential-limiting element or device geometry now to be described. Like many other attempts to meet the need, has not been commercialized.